1. Field of the Invention
The present invention uses N-acetylcysteine and levulose as cell protectors in the pharmacological therapy of many diseases that are followed by cell death associated with phenomena of oxidative stress.
2. Description of the Related Art
Various groups of scientists recently demonstrated that high concentrations of fructose prevent the anoxic death of hepatic cells. Various mechanisms were suggested to explain this protective effect:
a) an increment of the glycolytic production of lactate, which can directly protect the cells; b) an increased anaerobic synthesis of adenosine-triphosphate (ATP); c) an inhibition of the production of free radicals; d) a modulation of the homeostasis of Ca.sup.2+. In spite of these hypotheses, however, the molecular mechanisms involved in the protective effect afforded by the fructose are still not explained.
Under anaerobic conditions, levulose is a better glycolytic substrate than glucose or other carbohydrates and produces very high proportions of lactate. Just lactate (10 mH), however, does not prevent the anoxic death of cells. Thus, although lactate may contribute to the low intracellular pH (pH i) found after 30 minutes of anoxia in the presence of levulose, lactate by itself does not explain the protective function carried out by levulose against anoxic damage. The anoxic damage is clearly reduced by lowering the extracellular pH and, therefore, gives rise to an intracellular acidification. However, if the pH i is linked to an extracellular pH of 7.4 because of the use of monensine, which modulates the exchange of Na for H, levulose also protects against cell death. Therefore, both acidosis and levulose can protect against cell death by independent mechanisms.
In aerobic metabolisms, the necessary energy to maintain the cell integrity is supplied by the cytochromic mitochondrial system. In the case of anoxia, the lack of oxygen does not allow the formation of ATP by oxidative phosphorylation and the stored ATP is very quickly consumed. As a matter of fact, the depletion of ATP is a certain fact under hypoxic conditions or those of toxic damages. It is not clear, however, in which manner an ATP deficit becomes an irreversible damage. Levulose has shown to be effective in the protection of hepatocytes of rats against lethal damages caused by mitochondrial inhibitors, such as cyanide, oligomycin or menadione. Some authors suggested that the protective mechanism by levulose should entail the glycolytic production of ATP. Levulose, however, cannot protect the cells against the hypoxic damages by merely maintaining the glycolytic production of ATP.
An increased concentration of the free intracellular Ca.sup.2+, that can cause the activation of the degrading enzymes dependent on Ca.sup.2+, such as phospholipase A, endonuclease and protease, generally accompanies the necrosis of the liver. The function of the free intracellular Ca.sup.2+ in cytotoxicity was especially studied in the hepatocytes. Glutathione (GSH) plays a critical part in the regulating of the sequestrum of Ca.sup.2+ in the endoplasmic reticulum. Furthermore, under conditions of an increase in the forming of disulfide glutathione (GSSG), e.g., during an oxidative stress, the GSSG is reduced to GSH by the reductase glutathione, at the expense of nicotinamide-adenosine-dinucleotide-phosphate (NADPH). Therefore, the oxidation of GSH may facilitate a change in the amount of mitochondrial Ca.sup.2+.
Glutathione is always present in the eukaryotic cells and is involved in many cell functions. It is the most prevalent cellular thio and the most abundant peptide of low molecular weight present in the cells. The GSH acts as a reducing agent and as an antioxidant, serves as a reserve of cysteine, participates in reactions of detoxification of xenobiotics and in the metabolism of numerous cell compounds, it is required for the synthesis of some prostaglandins, and it is connected with the regulating of the cell cycle and with thermotolerance. This tripeptide plays a part in the protection against tissular damages resulting from the exposition to oxidant environments such as hyperoxia, hyperbaric oxygen or ozone, and it can also protect against radiations, ultraviolet light and photodynamic effects.
In view of the foregoing, a possible explanation of the protective effect of levulose with respect to the hepatic cell death, under toxic stress conditions, can be summarized in the hypothesis that there might exist a relationship between the maintaining of the levels of the cellular NADPH, the status of glutathione and calcic homeostasis, and the incidence of levulose on these cell phenomena.
On the other hand, various studies have shown the symptomatic amelioration of some diseases, e.g. chronic bronchitis, after treatment with N-acetylcysteine (NAC). In spite of some variations in the efficiency obtained between one and other studies, there are many data that suggest that the NAC can ameliorate the symptoms of chronic bronchitis. The most important improvements observed are with respect to the decrease in the amount and purulence of the sputum, the rate of exacerbation, and/or the number of sick days. Among the well known characteristics of bronchitis, mention must made of: ciliary paralysis, hyperplasia of the mucous cells, obstruction because of mucus, infection, inflammation, and cell lesion (fibrosis). In many instances, the beginning of bronchitis is caused by cigarette smoke. A vicious circle develops as a result of this lesion, that leads to hypoxia, emphysema and, oftentimes, death. From a clinical perspective, data exist that indicate that NAC may reduce the symptoms that weaken patients suffering from chronic bronchitis.
In order to explain the possible mechanisms that cause the symptomatic improvement of patients suffering from chronic bronchitis who had been treated with NAC, it must be taken into account: that on the one hand, it is probable that the mucolytic properties of NAC contribute to the clinical improvement; but that, on the other hand, it may happen that some of the antioxidant properties of NAC are also beneficial for patients suffering from chronic bronchitis. This last supposition is mainly based on the findings that iodoglycerol, a mucolytic not having any antioxidant properties, which reduces in an efficient manner some of the symptoms of bronchitis, does not seem to be as clinically efficient as NAC, which is a mucolytic with antioxidant properties.
Among the oxidative mechanisms that may contribute to the chronic bronchitis, it must be pointed out that, for example, tobacco smoke may alter the oxidant/antioxidant equilibrium in the lungs and that therefore it can influence the development of chronic bronchitis in various manners. Among the various possibilities that can produce alterations in the oxidant/antioxidant equilibrium are: a) an increase in the production or the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO), which leads to an increase in the forming of metabolites of oxygen in the pulmonary cells. In turn, the increased oxidative stress can increase the conversion of GSH to oxidized GSH (GSSG), inactivate the antiproteinases and/or bring about cellular hyperplasia. Likewise, tobacco smoke could increase the number and the activity of the alveolar macrophages or neutrophils in the lungs, and increase the release of oxygen radicals by said cells. Further, the oxygen radicals may form chemotaxis which, in turn, can attract more neutrophils. The attracted phagocytes could release more oxidants, as well as elastase, contributing even more to upset in a synergetic manner the oxidants/antioxidants equilibrium and to cause cell lesions.
Various studies conducted by different authors to examine the effect of NAC on the oxidants/antioxidants equilibrium revealed:
The alveolar macrophages (MA) obtained through the washing of the lungs of asymptotic cigarette smokers produce more superoxide anion in vitro than the MA of the lungs of healthy control individuals. PA1 The adjunction of neutrophils stimulated with forbol myristic acetate (PMA) produced lesions of the pulmonary epithelial cells cultivated in vitro, which is reflected by the release of Cr.sup.51 pre-incorporated into the epithelial cells. The treatment with NAC protected to a considerable extent against the lesion caused by neutrophils. PA1 The release of superoxide anion by the MA of cigarette smokers is reduced in the individuals who took NAC during an 8-week period. PA1 The number of cells of the high and low airways of rats exposed to cigarette smoke was reduced with NAC treatment. PA1 XO PA1 Phagocytes PA1 Mitochondrias PA1 AA metabolism PA1 Environmental pollution PA1 Tobacco PA1 SOD (dismutase superoxide) PA1 Catalase PA1 Vitamin E PA1 Ceruloplasmin PA1 Redux reactions of the glutathione
It can be definitively asserted that the oxidants/antioxidants equilibrium is a key element in many diseases, among them, chronic bronchitis.
A summary of the factors producing oxidant lesions and of those providing antioxidant defenses can be as follows:
Oxidant lesions are produced by:
Antioxidant defenses are:
NAC seems to possess the capacity of tilting the equilibrium in favor of the antioxidants, possibly acting as an efficient agonist of the GSH.
Those concerned with these and other problems recognize the need for an improved method in accordance with the present invention.